Horwitz A H, Heffernan L, Cass L, Miyada C G, Wilcox G
Mol Gen Genet. 1980;179(3):615-25. doi: 10.1007/BF00271752.
In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analyses demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%--3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araC mutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.
利用体内重组技术将大肠杆菌araBAD - araC基因的缺失片段克隆到杂种pBR322 - ara质粒上。遗传和物理分析表明，所需的缺失片段已重组到质粒上。除了能对各种ara缺失进行详细的物理分析外，该方法还产生了一系列质粒克隆载体，可用于通过体内重组克隆位于缺失片段所覆盖区域内的任何ara点突变。含有克隆点突变的杂种质粒可通过遗传互补与原始克隆载体区分开来。由于质粒ara区域与染色体ara区域之间的重组频率为0.025% - 3%，因此可以很容易地获得所需的重组质粒。利用一个缺失了ara控制位点区域和araC基因的质粒，通过体内重组克隆了两种类型的araBAD启动子突变和一个araC突变。对这些质粒的遗传和物理分析证实，所研究的突变已重组到ara缺失质粒上。本文讨论了该方法在ara基因及其他遗传系统中的应用。
